Comparison of Established Diagnostic Methodologies and a Novel Bacterial smpB Real-Time PCR Assay for Specific Detection of Haemophilus influenzae Isolates Associated with Respiratory Tract Infections

Kate Reddington, Stefan Schwenk, Nina Tuite, Gareth Platt, Danesh Davar, Helena Coughlan, Yoann Personne, Vanya Gant, Virve I. Enne, Alimuddin Zumla, Thomas Barry

H. influenzae is a significant causative agent of Respiratory Tract Infections (RTI) worldwide. There is a requirement for the development of a rapid H. influenzae diagnostics assay which would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients.A number of nucleic acid diagnostics approaches have been described in the literature that detect H. influenzae in RTIs, however there are reported specificity and sensitivity limitations for these assays. In this study a novel real-time PCR diagnostics assay targeting the smpB gene was designed to detect all serogroups of H. influenzae. The assay was validated using a panel of well characterised Haemophilus species.Subsequently, a collection of 44 Haemophilus clinical isolates were collected and 36 isolates were identified as H. influenzae using a gold standard methodology which combined the results of MALDI-TOF MS and a fucK diagnostics assay. Using the novel smpB diagnostics assay, 100% concordance was observed with the gold standard demonstrating a sensitivity of 100% (95% Confidance Interval (CI): 90.26-100.00) and a specificity of 100% (95% CI: 63.06 to 100.00) when used on clinical isolates.To demonstrate the clinical utility of the diagnostics assay presented, a panel of lower RTI samples (n =98) were blindly tested with the gold standard and smpB diagnostics assays. The results generated were concordant for 94/98 samples tested demonstrating a sensitivity of 90.91% (95% confidence interval [CI]: 78.33-97.47) and specificity of 100% (95% CI: 93.40 to 100.00) for the novel smpB assay when used directly on respiratory specimens.